There is evidence that secreted HIV-1 Nef plays an important role in affecting T cell function and in the survival of bystander uninfected cells in vivo. However, the mechanism(s) by which Nef is secreted has not been elucidated. We hypothesize that cellular proteins which interact with Nef regulate its secretion. Intracellularly, Nef is known to interact with the endosomal trafficking pathway, and has been shown to upregulate the exosomal machinery of the host cell. Furthermore, our group has mapped highly conserved Nef motifs in the N-terminal regions of both HIV and SIV Nef that are critical for secretion, and have engineered a peptide based on one of these Nef secretion motifs, the Secretion Modification Region (SMR). This SMRwt peptide, which contains only the SMR motif and which inhibits Nef vesicle secretion, was used to co-immunoprecipitate four cellular binding partners. One of these host cell proteins was identified as heat shock protein 70 family member, mortalin. Though much of the literature on mortalin centers on its role in blocking apoptosis through its interaction with the tumor suppressor protein p53, there is evidence that it is also involved in exosomal trafficking. As Nef also contains a p53-binding site in its N-terminal region, it is possible that the full-length Nef protein does not bind mortalin directly, but rather interacts with it via their mutual interactions with p53. We hypothesize that Nef and mortalin directly interact allowing Nef to catalyze the intracellular secretary process and release itself in exosomes-like vesicles. This is currently being tested through the following experiments, (i) FLAG-tagged mortalin is being used to co-immunoprecipitate wild type Nef-GFP from Jurkat cells to verify that, in addition to binding the SMRwt peptide, mortalin interacts with the full-length Nef protein;and, (ii) a binding assay with bacterial recombinant wild type Nef-GFP and purified FLAG-tagged mortalin is being used to determine if Nef interacts directly with mortalin, or indirectly via an unidentified host cell protein. Since the SMRwt peptide has been shown to inhibit Nef secretion, presumably by out-competing it for a cellular binding partner, we hypothesize that this inhibition is a result of disrupting mortalin's interaction with Nef. If true, mortalin is necessary for Nef secretion, and disrupting this interaction by other means will also inhibit Nef's secretion in exosome-like vesicles. To test this, the effect on Nef secretion will be assessed following (i) knockdown of the expression of mortalin using microRNA (miRNA);and, (ii) blocking of mortalin's interaction with Nef using antibody inhibition. The results of these experiments will generate fundamental knowledge of the cellular component(s) of Nef secretion. Given the proven difficulty in targeting Nef directly, this knowledge will open new therapeutic avenues to prevent AIDS pathogenesis through the newly identified host cell trafficking proteins. PUBLIC HEALTH RELEVANCE: The development of an HIV/AIDS vaccine has proven to be more difficult than anticipated, and the production of new antiviral drugs, many of them targeting viral proteins, has provided the greatest success in reducing AIDS deaths. This research will confirm the identification of a new set of cellular trafficking proteins involved in progression to AIDS, which could be potential therapeutic targets.